Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever in humans, continues to be a very significant health problem. Unlike other Salmonella enterica serovars, which can infect a variety of hosts and can cause self-limiting gastroenteritis (e. g. food poisoning), S. Typhi is an exclusive human pathogen where it causes typhoid fever, a life-threatening disease. It is estimated that there are ~20,000,000 cases of typhoid fever every year, resulting in 600,000 deaths. During the last funding period, we have focused our main efforts in the characterization of Typhoid toxin, a remarkable exotoxin discovered in our laboratory that is exclusively produced by and highly conserved in all S. Typhi and other typhoidal Salmonellae such as Salmonella Paratyphi. Typhoid toxin is a member of the so-called AB5 family of exotoxins, with a single B subunit and two A subunits, which are homologs of the A subunits of pertussis toxin and the Cytolethal Distending Toxin. The biology of Typhoid toxin is unique in that the toxin is produced only when bacteria are within mammalian cells. After its synthesis and assembly, the toxin is released into the Salmonella-containing vacuole and subsequently packaged into vesicle carriers that export the toxin to the extracellular medium, from where the toxin finds its way into target cells by interacting with specific surface receptors. During the last funding period we have characterized the composition of Typhoid toxin, solved its crystal structure, and obtained insights into the mechanisms by which it is secreted from the bacteria and transported to the extracellular environment. These studies have led to the discovery of a new mechanism of protein secretion in bacteria. We have also discovered the cellular receptor for Typhoid toxin, which has provided a mechanistic explanation for this toxin's broad cellular specificity, and implicated this toxin in the pathogenesis of typhoid fever, opening up a concrete path for the development of therapeutic and prevention strategies. Our studies have unexpectedly also led to major insights into the mechanisms of S. Typhi host specificity, uncovering what appears to be a novel microbial surveillance mechanism in macrophages that restricts S. Typhi growth in non-permissive species. Finally, we have developed a mouse model for S. Typhi infection by humanizing the mouse immune system with human stem cells. Thus, in a remarkably short period of time our efforts have provided major insights into arguably the most fundamental questions i Salmonella Typhi's research: the molecular bases for its human adaptation and host specificity, its ability to cause typhoid fever, and the development of an animal model to study it mechanisms to cause persistent infection. We intend to continue this line of investigation using the study of Typhoid toxin as a window into fundamental aspect of the mechanisms of S. Typhi pathogenesis. More specifically, we propose to study the mechanisms of Typhoid toxin secretion from the bacteria, its transport from the site of production within the Salmonella-containing vacuole to the extracellular space, and its interaction with its cellular receptors. We also plan to continue our efforts to improve a mouse model for S. Typhi infection.